Loss of MDA5 function does not affect replication of influenza virus or production of proinflammatory cytokines in respiratory epithelial cells. (A) Influenza (MOI: 0.1) transcripts, normalized to siNeg control at 24 h. Influenza-infected A549 cells were previously transfected with the indicated siRNA. (B) Immunoblotting showing efficiencies of MDA5, RIG-I, and MAVS protein expression, relative to HSP90 loading control, after transient transfection with the indicated siRNA into A549 cells. Transfected cells were left uninfected or infected with influenza strain A/Victoria/361/2011 (MOI: 0.5) for 24 h or treated with IFN-β (10 IU/ml) for 24 h. 20 µg of lysates were run per lane. Shown is a representative experiment corresponding to A. (C–F) Proinflammatory gene transcripts quantitated by qRT-PCR from A. Levels of IL-1α (C), IL-6 (D), IL-8 (E), and TNF (F) were normalized to β-actin and are shown relative to normalized levels at 8 h after infection. Data show means ± SD from six to seven experiments (A) and three independent experiments in (C–F). *, P < 0.05, by Kruskal-Wallis test (A); all other comparisons were nonsignificant.